Project Summary Salmonella enterica remains the major laboratory-confirmed pathogen of foodborne disease in the US and is responsible for typhoid fever in developing countries. As such, S. enterica infections incur substantial morbidity and mortality in both humans and animals worldwide. To identify new strategies that could reduce the virulence of S. enterica and support the development of alternative antimicrobials, our lab is focused on bacterial outer membrane vesicles (OMVs) that efficiently deliver inflammatory LPS to host cells with which they interact. OMVs from bacterial pathogens can modulate host cell signaling pathways that regulate innate immune responses and most recently have emerged as critical activators of the caspase-11-dependent non-canonical inflammasome response. Salmonella invade cells and immediately adapt to the lowered pH and magnesium levels of their intracellular vacuolar compartment by activating their two-component master regulator PhoPQ. In vitro growth conditions that recapitulate this environmental shift increase OMV production and modulate the repertoire of outer membrane proteins (OMPs) as OMVs bud from bacteria. We hypothesize that PhoPQ-dependent induction of distinct OMPs and/or LPS modifying enzymes will differentially regulate OMV number, size and OMP repertoire, thereby dictating host immune responses. In support, our preliminary data indicate that isolated OMVs from Salmonella grown under phoPQ-activating conditions induce a caspase 11-killing mechanism in BMDM. In this high risk/high impact exploratory R21, we will specifically focus on OMPs that may participate in OMV binding to host receptors to promote endocytosis (pagC, pagN, pgtE), lipidA-modifying enzymes (pagP, pagL, lpxO) and the activator of LPS-core decorating enzymes (pmrAB). Exciting new preliminary data identify both hyper- and hypo-vesiculating mutants in which the extent of OMV production directly correlates with the degree of caspase-11 activation and BMDM death. We shall continue to dissect the role of PhoPQ-regulated gene products in OMV phenotypes and determine how OMVs and their producing strains impact host cell death and/or inflammasome activation. Identifying Salmonella PhoPQ-regulated proteins that contribute to OMV biogenesis and OMV-dependent effects on cell death and innate immune responses will better define the pathogenesis of Salmonella and support the discovery of new targets for antimicrobial therapeutics. As OMV vaccines for other bacterial infections are already approved and on the market, and OMVs have also been proposed for use in targeted drug delivery, our findings will highlight OMV growth conditions and strain constructs that maximize immunogenicity while minimizing undesired side effects.